Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst. These cells possess the ability to differentiate into all cell types of the three germ-layers and to proliferate indefinitely. In defined conditions ES cells are committed to the mesodermal lineage and differentiate, amongst other cell types, into cardiomyocytes (CMs). The processes underlying mesodermal and subsequent cardiac differentiation are yet only partially understood. Catecholamine release is well known to modulate heart rate and force in adult mammals. Despite first evidence, only little is known about an involvement of catecholamines during embryonic heart development. Therefore the work aimed to investigate in more detail whether catecholamines are involved in the process of ES cell cardiac differentiation in vitro. Effects of catecholamine depletion induced by reserpine were investigated during murine D3 PIG44 ES cell differentiation. Reserpine is a drug blocking vesicular storage of monoamines, and as a result depletes cells of the catecholamines norepinephrine and epinephrine. Cardiac differentiation was assessed by quantification of beating clusters, immunocytochemistry, molecular biology, flowcytometry and pharmacological approaches. Proliferation and cytotoxicity was evaluated by embryoid body cross-section measurements and impedance monitoring, while functional characterization of CMs was performed using extracellular field potential (FP) recordings with microelectrode arrays (MEAs). Involvement of -adrenoceptor signaling was studied differentiating ES cells in the presence of reserpine and isoproterenol. To further discriminate between drug-specific effects of reserpine and catecholamine action via adrenergic receptors we applied the unspecific α- and β-receptor antagonists phentolamine and propranolol during differentiation. Reserpine treatment led to a remarkable reduction of beating cardiac clusters, down-regulation of cardiac proteins actinin and troponinT and delayed mesodermal and cardiac gene expression. In more detail, the average ratio of ~40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. In line, significant reduction by 71.6% (n=11) of eGFP expressing CMs after reserpine treatment was revealed by flowcytometry. Reserpine neither reduced EB size nor acted cytotoxic on CMs, while an increased numbers of neuronal cells were observed. MEA measurements with reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Co-application of isoproterenol and reserpine during differentiation partially rescued cardiac development. The developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine and proved an involvement of adrenergic receptors in the process. We conclude that catecholamines and adrenergic signaling play a critical role during cardiac development in ES cells. Since we experienced the ES cell/MEA model as useful for pharmacological approaches, we wondered as a next step whether pluripotent ES cell-derived CMs could be used for safety pharmacological drug screening with cardio-active compounds. Concentration/response was tested in rhesus monkey and human ES cell- and human induced pluripotent stem cell-derived CMs (rESCMs, hESCMs and, hiPSCMs, respectively) for different drugs at increasing concentrations. Empirical observation under physiologic positive and negative chronotropic drug-stimulation showed negative correlations of FP duration (FPDur) and beating frequency (negative FP/f correlation). Negative FP/f correlation persisted independent of cell lines in hESCMs and hiPSCMs. In contrast, FP/f correlations changed to positive values in the presence of E4031 and sotalol in a concentration-dependent manner. Therefore, it was concluded that safety pharmacological drug-screening is feasible using ES cell-derived CMs and MEAs independent of cell lines and species origin. Furthermore a novel method to qualitatively evaluate QT-time/repolarization prolonging drug effects based on FP/f correlations is sugested. The results may help to reduce cost-intensive clinical trials and, above all, to reduce the number of animal experiments.